Convection-Induced Biased Distribution of Actin Probes in Live Cells

Fluorescent markers that bind endogenous target proteins are frequently employed for quantitative live-cell imaging. To visualize the actin cytoskeleton in live cells, several actin-binding probes have been widely used. Among them, Lifeact is the most popular probe with ideal properties, including fast exchangeable binding kinetics. Because of its fast kinetics, Lifeact is generally believed to distribute evenly throughout cellular actin structures. In this study, however, we demonstrate misdistribution of Lifeact toward the rear of lamellipodia where actin filaments continuously move inward along the retrograde flow. Similarly, phalloidin showed biased misdistribution toward the rear of lamellipodia in live cells. We show evidence of convection-induced misdistribution of actin probes by both experimental data and physical models. Our findings warn about the potential error arising from the use of target-binding probes in quantitative live imaging.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1

Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration.     

Myosin II Motor Proteins with Different Functions Determine the Fate of Lamellipodia Extension during Cell Spreading

Non-muscle cells express multiple myosin-II motor proteins myosin IIA, myosin IIB and myosin IIC transcribed from different loci in the human genome. Due to a significant homology in their sequences, these ubiquitously expressed myosin II motor proteins are believed to have overlapping cellular functions, but the mechanistic details are not elucidated. The present study uncovered a mechanism that coordinates the distinctly localized myosin IIA and myosin IIB with unexpected opposite mechanical roles in maneuvering lamellipodia extension, a critical step in the initiation of cell invasion, spreading, and migration. Myosin IIB motor protein by localizing at the front drives lamellipodia extension during cell spreading. On the other hand, myosin IIA localizes next to myosin IIB and attenuates or retracts lamellipodia extension. Myosin IIA and IIB increase cell adhesion by regulating focal contacts formation in the spreading margins and central part of the spreading cell, respectively. Spreading cells expressing both myosin IIA and myosin IIB motor proteins display an organized actin network consisting of retrograde filaments, arcs and central filaments attached to focal contacts. This organized actin network especially arcs and focal contacts formation in the spreading margins were lost in myosin IIÂ cells. Surprisingly, myosin II$\widehat{B}$ cells displayed long parallel actin filaments connected to focal contacts in the spreading margins. Thus, with different roles in the regulation of the actin network and focal contacts formation, both myosin IIA and IIB determine the fate of lamellipodia extension during cell spreading.     

The fine structure of growing and non-growing whole glia cell preparations.

Human glia cells become blocked in G1 if starved of serum. The characteristics of the GI blocked state are flattening on the substrate, and absence of cell translocation, ruffling and macropinocytosis. Re-entry into the cell cycle, as a result of growth factor stimulation, is accompained and even preceded by the return of this cellular locomotion. We have studied the fine structure of intact human glia cells and ultrathin sections of these cells when proliferating normally in vitro, when starved of serum and during their return to the cell cycle following stimulation with mEGF (mouse epidermal growth factor). Particular attention was paid to morphologically definable components of the cellular musculoskeletal system. Proliferating interphase glia generally had a leading lamella containing few organelles and oriented bundles of 7 nm microfilaments with structureless lamellipodia at their tips, which often formed ruffles. The perinuclear area was thick and contained many cell organelles, including mitochondria and secondary lysosomes. Glia starved of serum were thinly spread; their peripheral cytoplasm was filled with a diffuse mat of microfilaments, they had no structureless lamellipodia and their perinuclear areas, although thinner, contained cell organelles in equal amounts and of similar type of those found in proliferating cells. On EGF stimulation, after approximately 2 hours the perinuclear area of the cells thickened, and structureless lamellipodia subsequently appeared at the tips of the leading lamellae, forming ruffles. The cells finally began to translocate, the process being accompained by the reorientation and packing of the microfilaments into bundles. As the kinetics of EGF binding and break down by glia cells are similar to those described for fibroblasts, the findings do not support the concept of EGF receptor interactions inducing ultrastructurally demonstrable microfilament or other musculoskeletal structural changes in the cell. They do, however, define the differing cellular morphologies of motile and immobile structures.     

How to measure Ca2+ in cellular organelles?

The review will aim to briefly summarise information on calcium measurements in cellular organelles with emphases on studies conducted in live cells using optical probes. When appropriate we will try to compare the effectiveness of different indicators for intraorganellar calcium measurements. We will consider calcium measurements in endoplasmic reticulum, Golgi apparatus, endosomes/lysosomes, nucleoplasm, nuclear envelope, mitochondria and secretory granules.     

Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP-actin fusion protein

Directional migration of border cells in the Drosophila egg chambers is a developmentally regulated event that requires dynamic cellular functions. In this study, the electron microscopic observation of migrating border cells revealed loose actin bundles in forepart lamellipodia and numerous microvilli extending from nurse cells and providing multiple adhesive contacts with border cells. To analyze the dynamics of actin in migrating border cells in vivo, we constructed a green fluorescent protein-actin fusion protein and induced its expression in Drosophila using the GAL4/UAS system. The green fluorescent protein-actin was incorporated into the actin bundles and it enabled visualization of the rapid cytoskeletal changes in border cell lamellipodia. During the growth of the lamellipodia, the actin bundles that increased in number and size radiated from the bundle-organizing center. Quantification of the fluorescence intensity showed that an accumulation of bundle-associated and spotted green fluorescent protein-actin signals took place during their centripetal movement. Our results favored a treadmilling model for actin behavior in border cell lamellipodia.     

Three-dimensional study of epithelioid cells by a quick-freezing and deep-etching method in muramyl dipeptide-induced granulomas

The three-dimensional ultrastructure of epithelioid cells was studied by the quick-freezing and deepetching (QF-DE), as well as the freeze-substitution (QF-FS) methods. The granulomas were induced in rats by injecting muramyl dipeptide (MDP) into the hind footpads. At 3 weeks after the injection, the footpads were perfused with a fixative, excised, and quickly frozen to prepare the replica membranes. Some unfixed footpads were also quickly frozen and freeze-substituted. Dense networks of intermediate filaments, connected with the nuclei, mitochondria and other vesicular cell organelles, were observed throughout the cytoplasm of epithelioid cells by the QF-DE method. A few actin filaments were located in filopodia and just beneath the cell membranes. Interdigitation of the cell membranes between adjacent cells was clearly demonstrated by the QF-FS method and clathrin-coated pits were identified at the base of interdigitating filopodia. In addition, the exact moment of fusion between endosomes and lysosomes was ascertained by the same method. These results suggest that the cytoskeletal organization of epithelioid cells resembles that of epithelial cells rather than actively motile macrophages.     

Three-dimensional study of epithelioid cells by a quick-freezing and deep-etching method in muramyl dipeptide-induced granulomas.

The three-dimensional ultrastructure of epithelioid cells was studied by the quick-freezing and deep-etching (QF-DE), as well as the freeze-substitution (QF-FS) methods. The granulomas were induced in rats by injecting muramyl dipeptide (MDP) into the hind footpads. At 3 weeks after the injection, the footpads were perfused with a fixative, excised, and quickly frozen to prepare the replica membranes. Some unfixed footpads were also quickly frozen and freeze-substituted. Dense networks of intermediate filaments, connected with the nuclei, mitochondria and other vesicular cell organelles, were observed throughout the cytoplasm of epithelioid cells by the QF-DE method. A few actin filaments were located in filopodia and just beneath the cell membranes. Interdigitation of the cell membranes between adjacent cells was clearly demonstrated by the QF-FS method and clathrin-coated pits were identified at the base of interdigitating filopodia. In addition, the exact moment of fusion between endosomes and lysosomes was ascertained by the same method. These results suggest that the cytoskeletal organization of epithelioid cells resembles that of epithelial cells rather than actively motile macrophages.     

Co-clustering of Golgi complex and other cytoplasmic organelles to crescentic region of half-moon nuclei during apoptosis

Early apoptosis is defined by stereotypic morphological changes, especially evident in the nucleus, where chromatin condenses and compacts, and assumes a globular, half-moon or crescent-shaped morphology. Accumulating evidence suggests that cytoplasmic organelles such as mitochondria and the Golgi complex are major sites of integration of pro-apoptotic signaling. In this study, cytoplasmic organelles including Golgi complex, mitochondria, endosomes, lysosomes, and peroxisomes were shown to condense at the same unique region adjacent to the crescentic nucleus during a relatively early stage of apoptosis induced by staurosporine or other agents. The co-clustering phenomenon may be caused by shrinkage of cytoplasm during apoptosis although cytoskeletal markers actin and tubulin were not condensed and appeared excluded. These data suggest the co-clustering of cytoplasmic organelles plays an interesting role during the progression of the apoptotic process. It is possible that modification of pro-apoptotic proteins may arise as a result of the interplay of these cytoplasmic organelles.     

mEosFP-based green-to-red photoconvertible subcellular probes for plants

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.     

Maintaining social contacts: The physiological relevance of organelle interactions

Membrane-bound organelles in eukaryotic cells form an interactive network to coordinate and facilitate cellular functions. The formation of close contacts, termed “membrane contact sites” (MCSs), represents an intriguing strategy for organelle interaction and coordinated interplay. Emerging research is rapidly revealing new details of MCSs. They represent ubiquitous and diverse structures, which are important for many aspects of cell physiology and homeostasis. Here, we provide a comprehensive overview of the physiological relevance of organelle contacts. We focus on mitochondria, peroxisomes, the Golgi complex and the plasma membrane, and discuss the most recent findings on their interactions with other subcellular organelles and their multiple functions, including membrane contacts with the ER, lipid droplets and the endosomal/lysosomal compartment.     

Alternative modes of organellar segregation during sporulation in Saccharomyces cerevisiae

Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.     

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.     

Changes in fused cells induced by hvj (sendai virus): redistribution of cytoplasmic organelles and cytoskeletal reorganization

To understand the relationship between the location of organelles and cellular function, we examined the dynamic state of cytoplasmic organelles and cytoskeleton in polynuclear Ehrlich ascites tumor (EAT) cells fused with hemagglutinating virus of Japan (HVJ; Sendai virus) by confocal laser scanning microscopy. Irregular fused cells gradually became spherical during culture, and nuclei and mitochondria were redistributed in the fused cell; nuclei formed a cluster surrounded by mitochondria. F-actin, vimentin, and microtubules were also reorganized with the redistribution of cell organelles. Further, when the morphological change was inhibited by L4-1, a chlorophyll-like substance derived from silkworm faeces, or pyropheophorbide-a, the arrangement of organelles and cytoskeleton remained disturbed, suggesting that the movement of the cytoskeleton is closely associated with cell shape and the distribution of cytoplasmic organelles.     

RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells

RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.     

Glycogen synthase kinase-3 regulates cytoskeleton and translocation of Rac1 in long cellular extensions of human keratinocytes

Wound keratinocytes form long cellular extensions that facilitate their migration from the wound edge into provisional matrix. We have previously shown that similar extensions can be induced by a long-term exposure to EGF or rapidly by staurosporine in cultured cells. This morphological change depends on the activity of glycogen synthase kinase-3 (GSK-3). Here, we have characterized the cytoskeletal changes involved in formation of these extended lamellipodia (E-lam) in human HaCaT keratinocytes. E-lams contained actin filaments, stable microtubules and keratin intermediate filaments. E-lam formation was prevented by cytochalasin D, colchicine and low concentrations of taxol and nocodazole, suggesting that actin and microtubule organization and dynamics are essential for E-lam formation. Staurosporine induced recruitment of filamentous actin (F-actin), cortactin, filamin, Arp2/3 complex, Rac1 GTPase and phospholipase C-gamma1 (PLC-gamma1) to lamellipodia. Treatment of cells with the GSK-3 inhibitors SB-415286 and LiCl(2) inhibited E-lam formation and prevented the accumulation of Rac1 and Arp2/3 complex at lamellipodia. The formation of E-lams was dependent on fibronectin-binding integrins and normally regulated Rac1, and expression of either dominant-negative or constitutively active forms of Rac1 prevented E-lam formation. Overexpression of either RhoA or Cdc42 GTPases suppressed E-lam formation. We conclude that extended lamellipodia formation in keratinocytes requires actin and tubulin assembly at the leading edge, and this process is regulated by Rac1 downstream of GSK-3.     

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.     

Redundancy of lamellipodia in locomoting Walker carcinosarcoma cells

Locomoting metazoan cells usually form lamellipodia at the leading front and it is widely accepted that lamellipodia are required for locomotion. In this case, suppression of lamellipodia must stop locomotion. However, the experiments show that lamellipodia are redundant for locomotion of Walker carcinosarcoma cells. Low latrunculin A concentrations (10(-7) M) transform polarised locomoting cells with lamellipodia into cells without morphologically recognisable protrusions showing an increased speed of locomotion and a reduced amount of cellular F-actin. Whereas untreated cells show a fairly linear distribution of F-actin along the plasma membrane, cells lacking morphologically recognizable protrusions at the front show modifications at the front consisting in an irregular distribution of F-actin with formation of small or large patches of F-actin alternating with small or large gaps in the F-actin layer. This is associated with a reduced resistance to deformation pressure at the front of the cell. High concentrations of latrunculin A (>10(-7) M) compromising contraction at the rear stop locomotion, suggesting that cortical contraction is important for locomotion to occur in these cells. The results are consistent with the view that actin polymerization is important for formation of lamellipodia but they are not compatible with the view that lamellipodia are essential for locomotion of Walker carcinosarcoma cells. A unifying hypothesis for the formation of different types of protrusions is proposed.     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.